1. Overview of Existing Single-Cell Sequencing Technology
1) At present, single-cell sequencing technology is mainly used for the study of the development of human or mammalian embryonic cells and stem cells. Such cells only have cell membranes and have no cell walls, so it is not necessary to break the walls, and is relatively easy to extract gDNA from trace cells.
2) It is reported that single-cell sequencing technology is used for the study of plant seed development and cell sequencing of plant endophytic fungi. Such cells can be isolated and cultured, so it is relatively easy to obtain pure cell lines for molecular detection experiments.
2. Overview of Existing Fungal Wall-Breaking and DNA Extraction Technologies
1) Physical method: liquid nitrogen is added into a mortar for grinding, or a grinder is used for a low-temperature grinding. However, a large number of fungi are required in this method, thus the method cannot be applied to trace samples.
2) Chemical method: benzyl chloride extraction method, etc. The extracted DNA is poor in integrity and degrades easily. Generally, the DNA extracted by this method is only enough for PCR amplification, it is difficult to achieve the integrity requirements for the use in the construction of the gene libraries.
3) Enzymatic method: snail enzyme, cellulase, lysozyme, lywallzyme, etc. This method has low efficiency, and the amount of DNA extracted is not enough for trace samples to construct gene libraries.
3. Overview of Existing Detection Techniques for Trace Fungal Samples:
1) Microscopic examination: Direct observation under the microscope. Although the method is simple and fast, it has low sensitivity and some fungi may get missed easily, thus requiring rich operational experience.
2) Culture: The morphology and biochemical indicators of fungi in the culture process can be used as important standards for detection, but it is time-consuming and can get contaminated easily, and many fungi (especially pathogenic fungi) are difficult to be successfully cultured under artificial culture conditions.
3) PCR and gene chip technology: It is suitable for fungal detection with a clear range, which requires to design the primers and probes specifically, and this technology is not suitable for completely unknown species.
4) First-generation sequencing after PCR amplification using universal primers: It is suitable for the detection of single unknown fungi, generally applicable for detection of cultivable pure fungi, and is not suitable for detection of complex samples at one time.
5) Application of next-generation metagenome sequencing: It can be used for the detection of complex samples. However, since it is difficult to extract the DNA of fungi as compared to that of other microorganisms or mammals, a large amount of data irrelevant to the target species will be generated, making it very difficult to identify the data.
According to the above overviews of the prior art, it can be seen that the current detection techniques for trace fungi mainly have the following technical disadvantages.
1. It is difficult to perform efficient wall-breaking on trace fungi, non-cultivable fungi or difficult-to-culture fungi.
2. It is difficult to extract a sufficient amount of gDNA that can be used for the construction of gene libraries from trace fungi.
3. It is difficult to detect specific fungi in complex samples.
In conclusion, the extraction of complete genomic DNA by breaking the fungal wall has been a difficult problem in the field, especially for trace fungal samples. How to extract the fungal gDNA to reach the amount required for library construction for the second-generation sequencing from trace complex samples, and to perform the whole genome sequencing on the fungal gDNA have not been reported yet.
The method of the present invention can well overcome the above-mentioned technical defects by performing the steps of performing wall-breaking treatment on trace fungal cells, extracting gDNA, amplifying the gDNA, constructing the libraries for next-generation sequencing and performing genome sequencing and analysis, thereby realizing the high-efficiency detection of the trace fungi. The method of the present invention can be directly applied to the isolation, detection, identification of trace fungal samples, difficult-to-identify fungal samples or mixed samples, and in-depth study of genetic information. The fungi detection kit prepared according to the method of the present invention can be widely applied in various fields such as industrial production, environmental monitoring, air testing, soil testing, water quality testing, food testing, drug testing, cosmetics testing, health care products testing and medical testing, etc. The popularization and application of the technology of the present invention will have a good market prospect and generate considerable economic and social benefits.